SPIM microscope with a sequential light sheet

ABSTRACT

A SPIM-microscope (Selective Plane Imaging Microscope) having a y-direction illumination light source and a z-direction detection light camera. An x-scanner generates a sequential light sheet by scanning the illumination light beam in the x-direction. The SPIM-microscope has an illumination optics having a zoom optics provided in a beam path of the illumination light beam, the zoom optics being adapted to change the focal length of the illumination light beam and adapted to detect a larger area of the object by sequentially detecting sequences of images along the y-direction that have an increased resolution along the z-direction. An image processing unit combines these sequences of images by image stitching into one large overall image.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of the U.S.non-provisional application Ser. No. 16/385,883, which is a Continuationapplication of the U.S. non-provisional application Ser. No. 14/727,570,which is a Continuation application of the U.S. non-provisionalapplication Ser. No. 13/278,986 that claims the priority of the Germanpatent application DE 102010060121.7 having a filing date of Oct. 22,2010 and claims priority of the European patent application EP11169989.8 having a filing date of Jun. 15, 2011. The entire content ofthis prior German patent application DE 102010060121.7 and Europeanpatent application EP 11169989.8 and of the parent application Ser. Nos.16/385,883, 14/727,570 and 13/278,986 is herewith incorporated byreference.

BACKGROUND OF THE INVENTION

The invention relates to a SPIM-microscope comprising a light sourcesending an illumination light beam from a y-direction onto an object tobe imaged and a camera detecting in a z-direction as a first detectiondirection light emanating from the object as fluorescent light and/or asreflected light, wherein the z-direction extends substantiallyperpendicular to the y-direction.

In particular, biological samples should be analyzed both quickly andwithout damaging the sample. For many applications, it is useful togenerate a 3-dimensional image. Scattering artifacts and absorptionartifacts should be avoided that may occur due to interaction of theillumination light with the sample, in particular in the field offluorescence microscopy where the illumination light has the function ofan excitation light for exciting fluorescence.

For analyzing microscopic samples fast, without causing damage and witha high resolution the so-called SPIM technology is specifically suitable(Selective Plane Illumination Microscopy) where the illumination lightgenerates a light sheet, while the detection light generated byfluorescence and reflection is detected in a perpendicular directioncompared to the illumination direction by a camera.

A light sheet is an illumination volume with a substantially rectangularcross-section that is very thin in a first cross-sectional direction(here the z-direction) and significantly larger in a secondcross-sectional direction (here the x-direction) in comparison to thefirst cross-sectional direction. The illumination direction (here they-direction) extends substantially perpendicular to the firstcross-sectional direction (here the z-direction) and substantiallyperpendicular to the second cross-sectional direction (here thex-direction). The light sheet is focused by a cylindrical lens and thefocus or a focal length of the light sheet is to be understood as acertain range that extends in the illumination direction (here they-direction) where the light sheet is particularly thin so that theilluminated volume has the shape of a sheet, i.e. is very thin in thez-direction and much larger in the x- and in the y-direction.

Generating a light sheet according to the prior art SPIM technologyusing a cylindrical lens has the disadvantage that the system is quiteunflexible, for instance provides a fixed focus and therefore apredetermined illuminated volume. For achieving a high resolution, avery thin and long focus is advantageous. This focus can be scanned forobtaining a 3-dimensional image in one direction over the sample. Sincean increased length also increases the width the resolution in thez-direction is decreased. This means that a long focus at a lownumerical aperture of the illumination optics has the consequence thatalso the thickness of the illuminated volume is high. This means thatthe optical resolution along the optical axis in the detection directionis likewise low. By interaction of the excitation light with the samplescattering artifacts and absorption artifacts are generated which arevisible as striations or shadows in the image along the illuminationaxis, which is also referred to as “Curtain-Effects”.

One prior art approach for reducing the curtain-effects is themSPIM-technology according to which in addition in the telecentricarrangement by means of a resonant mirror the light sheet is tilted inrelation to the optical axis so that the illumination light beamincidents from a variety of direction's onto the sample, resulting inreducing the scattering artifacts and absorption artifacts. In simpleterms, varying the incident direction provides some backgroundillumination for the absorbing areas within the sample so that thestriations or shadows in the image are reduced. A disadvantage of thistechnology is the additional complexity, in particular if the lightsheet should be scanned in the z-direction for generating a3-dimensional image. Apart from that, this still does not solve theproblem of the low flexibility due to the predetermined focus.

SUMMARY OF THE INVENTION

It is an object of the invention to increase the flexibility of themicroscope described at the outset and at the same time achieve a higherresolution and detect more image data.

According to the invention, this is achieved by a SPIM-microscopecomprising: a light source sending an illumination light beam from ay-direction onto an object to be imaged; a camera detecting in az-direction as a first detection direction light emanating from theobject as at least one of fluorescent light and reflected light, whereinthe z-direction extends substantially perpendicular to the y-direction;an x-scanner generating a sequential light sheet by scanning theillumination light beam in an x-direction, wherein the x-directionextends substantially perpendicular to the y-direction and to thez-direction and the light sheet is sequentially formed in a plane thatis defined by the x-direction and the y-direction; an illuminationoptics comprising a zoom optics provided in a beam path of theillumination light beam, the zoom optics being adapted to change thefocal length of the illumination light beam and adapted to detect alarger area of the object by sequentially detecting sequences of imagesalong the y-direction that have an increased resolution along thez-direction; and an image processing unit that combines these sequencesof images by image stitching into one large overall image.

DETAILED DESCRIPTION OF THE INVENTION

According to a preferred embodiment of the invention, a photodetector isprovided detecting detection light sent from the object in the oppositedirection of the y-direction as a second detection direction, thedetection light being fluorescent light and/or reflected light. Thisgenerates a second data stream that can for instance be used in additionfor generating an image confocally. This optional additional image canbe switched on or off as desired.

When using multiphoton lasers, confocal detection as well as thenon-descanning light path can be chosen for generating images bymultiphoton detection. The non-descanning detection path has theadvantage of providing a higher collection efficiency, specifically incase of thick samples. The term non-descanning should be understood ascoupling out detection light directly at the detection objective.

If the SPIM detection beam path (in z-direction) is used for collectingthe fluorescent light and the light is a directed into a photomultiplieror an APD or APD array, a much higher efficiency is achieved than byusing the illumination optics for collecting the fluorescent light sincetypically the used numerical aperture of the illumination system islower than the numerical aperture of the detection system of the SPIMarrangement.

According to a further preferred embodiment of the invention, inparallel to the 2-dimensional wide field image generated by theSPIM-technology also confocally, optionally by using a multiphotonillumination, a 1-dimensional image of the object is generated thatcomprises a line extending in the x-direction. In this case, the2-dimensional image images that illumination layer that is illuminatedby the light sheet and as described above is influenced by the zoomoptics, while in parallel a so-called x-t-image is generated, that meansa line image that may in particularly in biological samples provideinformation about the velocity of movement of specific elements withinthe object, for instance provide information about the diffusion ofmolecules or other cell parts (organelles).

Preferably, for generating a 3-dimensional image, a first z-scanner isprovided moving the object in the z-direction so that sequentially aplurality of light sheets spaced in z-direction with respect to eachother are generated in the respective illumination planes, wherein adistance between the respective light sheets and the camera remainsunchanged. An advantage is that the entire illumination optics as wellas the SPIM detection optics and the confocal detection optics that canoptionally be switched on in addition can remain at the same locationand in addition the illumination beam does not need to be deflected inthe z-direction by a scanner, therefore simplifying the illuminationoptics. In the alternative, it is also possible to move the illuminationoptics or to deflect the illumination beam, for instance by means of anAcousto Optical Deflector (AOD) or by means of a galvanometer. In thiscase, it is preferable to have the SPIM detection optics trackingbehind, which can be accomplished by changing the position of theobjective of the SPIM detection optic or in the alternative by movingthe entire SPIM detection optics including the camera.

According to another preferred embodiment of the invention, an imageprocessing unit is provided that combines the sequentially generatedimages correlating to the respective plurality of light sheets which arespaced in the z-direction in a distance to each other to a 3-dimensionalimage.

According to another preferred embodiment of the invention, in parallelto the 3-dimensional image generated by the SPIM-technology alsoconfocally a 2-dimensional image of the object is generated in a planedefined by the x-direction and the z-direction.

According to another preferred embodiment of the invention, a secondz-scanner is provided moving the illumination light beam in thez-direction in relation to the object for generating sequentially aplurality of light sheets spaced in the z-direction with respect to eachother, and a third z-scanner is provided that tracks the detection beampath according to the deflection of the illumination light beam inz-direction by the second z-scanner so that a distance between the lightsheet and the camera remains unchanged. The data stream generated inthis fashion is preferably processed by an image processing unit thatcombines the sequentially generated images correlating to the respectiveplurality of light sheets which are spaced in the z-direction in adistance to each other to a 3-dimensional image by a variety of possibleRendering methods (Projection, Transparent, Shading, Ray Tracing etc.).Preferably, in addition to the 3-dimensional image that is generated byrendering, in parallel to the 3-dimensional image generated by theSPIM-technology also confocally (multiphoton) a 2-dimensional image ofthe object is generated in a plane defined by the x-direction and thez-direction.

If a larger area of the object should be imaged, this can beaccomplished by so-called “Stitching”, i.e. combining the sequentiallyimaged adjacent object areas. For this purpose, the object is moved inthe x-direction and a new area is illuminated and the image detected bya camera. The number of movements in the x-direction depends on the sizeof the object, the size of the field of the objective, and the cameraparameters. It is also possible to move the object in the y-directionfor detecting a larger area of the object. All of these images can becombined into one large overall image.

By a flexible optical zoom arrangement the size of the light sheet (inthe y-direction and in the z-direction) can be adjusted.

If it is desired to illuminate only a thin layer, the numerical apertureof the zoom optics is increased, having the consequences of decreasingthe usable size of the light sheet.

By sequentially detecting images along the y-direction (Stitching)sequences of images can be detected that have an increased resolutionalong the z-direction.

Since the optical parameters of the zoom optics are known, by choosingsuitable image processing only those areas can be used and combined thatprovide the increased resolution.

The zoom optics allowing varying the focal length of the illuminationlight beam can be an optical zoom according to a preferred embodiment ofthe invention, having lens groups which are mechanically moved withrespect to each other. By the zoom optics in combination with theillumination objective the focal length of the illumination light beamcan be expanded or shortened by changing the numerical aperture,allowing expanding or shortening the length of the field that isilluminated by the light sheet in the y-illumination direction.

According to another preferred embodiment, an electronic zoom isprovided that is adapted to change the scanning length in thex-direction. Preferably, the number of image pixels in x-direction ismaintained unchanged by the electronics zoom independently of thescanning length in x-direction that has been selected. If the scanninglength in the x-direction is reduced, but the number of scanned pixelsis kept unchanged over that shorter scanning length, the resolution canbe increased in case of a confocal detection up to reaching the NyquistTheorem.

If simultaneously a confocally detected image is generated, it is notedthat also this image comprises a higher resolution up to reaching theNyquist Theorem in the x-direction but at a smaller size of the image inthe x-direction, which has the function of an electronic zoom.

According to a preferred embodiment of the invention, an electronic zoomis provided in addition by which the scanning length in the z-directioncan be changed along which a plurality of spaced apart light sheets aregenerated sequentially. In this case, substantially the same as for theelectronic zoom acting in x-direction applies. Preferably, the number oflight sheets which are spaced with respect to each other in thez-direction is maintained unchanged by the electronics zoomindependently of the scanning length in the z-direction that has beenselected.

According to another preferred embodiment, the illumination light beamscanned in the x-direction is turned off at or close to the return pointwhere the maximum scanning length in x-direction has been reached. Thisallows avoiding damage to the sample since less light is sent onto thesample at the return points. In addition, this also avoids that theimage appears to be specifically bright at the return points since theillumination is maintained for a longer period of time at the returnpoints in comparison to other areas of the sample. In the ideal case,the scanning velocity in x-direction follows a saw-tooth graph. If thescanning velocity is very high, this is, however, difficult toaccomplish so that instead a sinus graph is chosen, resulting at thereturn points in a slower scanning velocity than in the middle of thescanning path. In particular for sinus curves it is useful if theillumination beam is turned off near the return points.

According to another preferred embodiment of the invention, the camerais an area detector chosen from the group consisting of CMOS-camera,CCD-camera or array-detectors. Since the SPIM technology is a wide fieldmicroscopy technology, the camera should provide a localization of thedetection light.

According to another preferred embodiment of the invention, themicroscope according to the invention is provided with a switch adaptedto switch between the following operational modes: confocal detection ofdetection light opposite to the y-illumination direction; SPIM-detectionof wide field detection light in the z-direction; and simultaneousdetection of the aforementioned confocal detection and SPIM-detection.

According to another preferred embodiment of the invention, adeactivation light source is provided sending from the y-direction adeactivation light beam onto the object making the sequentiallygenerated light sheet in the z-direction thinner, wherein thedeactivation light beam is sent offset in the z-direction onto theobject in relation to the illumination light beam and extends inparallel to that illumination light beam that is scanned in thex-direction. Preferably, the cross-section of the deactivation lightbeam has been modulated such that it comprises two maxima that areprovided as is viewed in the z-direction in front and behind the centerof the excitation beam in which center the excitation beam has a zeropoint in between the maxima. It is, however, in the alternative alsopossible to send two separate laser beams as deactivation beams, or tomake the light sheet only on one side thinner, i.e. as viewed in thez-direction, to provide only one deactivation beam in front of theexcitation beam or behind the excitation beam.

According to another preferred embodiment of the invention an excitationlight beam modulator is provided that is adapted to modulate theexcitation light beam into a Bessel beam. A Bessel beam comprises aninhomogeneous intensity distribution over the beam cross-section,comprising a relatively sharp main maximum and several significant sidelobes. In particular when adding a STED deactivation beam, the sidelobes can be suppressed by deactivation, so that only a narrow mainmaximum in the center of the excitation beam remains. A Bessel beamfurther has the advantage to reform behind a relatively opaque sectionat least partially so that curtain-effects are decreased further.

According to another preferred embodiment of the invention, theillumination light source is a pulsed laser; the illumination light beamis a multiphoton laser beam; and a multiphoton signal is detected in thez-direction. The specific advantage is that the SPIM detection opticshas a bigger numerical aperture and therefore provides for a highersignal strength detection for detecting the multiphoton signal.

According to another preferred embodiment of the invention the camera isa fast camera that is adapted to detect in addition to the SPIM signalalso the multiphoton signal. Fast cameras can for instance detect up to1000 images per second in a format 512×512.

According to another preferred embodiment of the invention, a switchablemirror is provided, allowing extracting the multiphoton signal from thez-direction and directing it to a photodetector. Photodetectors aregenerally faster than cameras, but provide no localization, which,however, is not absolutely necessary if a localization can be providedvia the multiphoton illumination beam, which is in particular easy toaccomplish in case of a multiphoton illumination since for this type ofillumination it is known which small volume within the object isilluminated at a specific point in time, so that for detecting thesignal the light coming from all direction scan be detected.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is discussed in the following by referring to thedrawings. In the drawings show:

FIG. 1 a schematic perspective view of the basic principle of theSPIM-technology according to the prior art;

FIG. 2 a schematic view of the illumination beam paths and the detectionbeam paths according to the SPIM-technology according to the prior art;

FIG. 3 a schematic view of the microscope according to the invention;

FIG. 4 a schematic view of the microscope according to the invention asshown in FIG. 3 but viewed in the x-scanning direction, including theillumination beam path and the detection beam path;

FIG. 5 a schematic view of the microscope shown in FIG. 4 , but viewedin the y-illumination direction;

FIG. 6 a schematic view that also demonstrates the STED-principleaccording to the prior art;

FIG. 7 is schematic view demonstrating the modulation of theSTED-deactivation beam by a phase plate;

FIG. 8 a the cross-section of an excitation beam as well as the relatedintensity distribution with the coordinates x, y and z;

FIG. 8 b the excitation beam plus the deactivation beam compared to eachother as well as the sequential light sheet generated by scanning inx-direction together with the related intensity distribution with thecoordinates x, y and z.

FIG. 9 a schematic view of an additional embodiment of the invention,using the SPIM-detection optics in addition for detecting a multiphotonsignal.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in a simplified fashion the principle of a SPIM-microscopeaccording to the prior art, operating based on the “Selective PlaneImaging” technology principle. Illumination is performed in they-direction by a light sheet for illuminating the object in a specificobject plane. The detection direction extends substantiallyperpendicular to the light sheet 1, i.e. in z-direction, wherein thedetection of the detection light is performed by an objective 3. Asshown in FIG. 1 , the light sheet 1 should be specifically thin over arange of the inner section for obtaining a high a resolution in thez-direction. A high resolution is accomplished if the light sheet 1comprises a narrow focus within the object.

As will be explained below in more detail, the length of this focus canbe influenced, making the imaged field larger, but decreasing thesharpness of the focus and therefore decreasing the narrowness of thelight sheet 1 and therefore decreasing the resolution of the picture inthe z-direction. Depending on the specific application, it might beuseful to have less resolution in the z-direction but at the same timeview a larger field and a larger imaging volume based on a thicker lightsheet. A larger imaging volume might also be useful if the generatedimage still allows to view the aspects of interest of the image well,but at the same time allows for a larger imaging volume having theadvantage that it is easier to ascertain that the imaged volume doesindeed contain the aspect of interest. If an image of an even higherresolution should then be generated of the aspect of interest it ispossible to manipulate the focus of the sequential light sheet 1 by thezoom-optics to make the focus smaller but sharper.

In contrast to confocal scanning microscopy, detection of the detectionlight in the z-direction according to the SPIM-technology requireslocalization of the detected light since the SPIM-technology is awide-field microscopy technology. The localization is typicallyaccomplished by a camera, for instance a CCD-camera or a CMOS-camera. Ifa 3-dimensional image of an object should be generated by theSPIM-technology, the light sheet 1 can be scanned in z-direction and theimages obtained in the various illumination planes can be combined togenerate a 3-dimensional image. This image processing is also named“Rendering”, in this case in the z-direction.

FIG. 2 shows schematically an illumination beam path according to theprior art in a SPIM-microscope. A laser 4 generates an illumination beam5 that is sent through a beam expander into the collimating lens 6 thatis succeeded by a cylindrical lens 7 focusing the light sheet 1 onto theobject. An objective lens collects the detection light and directs itinto a camera 9. The focus of the light sheet can be influenced bymoving elements in the group of lenses, in this strongly simplifiedexample according to FIG. 2 by moving the cylindrical lens 7 in relationto the collimating lens 6.

FIG. 3 shows the schematic structure of the SPIM-microscope according tothe invention, having an illumination beam path denoted by referencenumeral 10 and a SPIM-detection light beam path denoted with thereference 11. The illumination optics beam path 10 comprises in additionthe function of a confocal detection light beam path, but extending inthe opposite direction compared to the illumination direction.

First of all, the illumination optics beam path is described; a laser 4generates illumination light that is sent via a scanner 12 into the zoomoptics 13. The scanner 12 generates a sequential light sheet 1 forilluminating the object 2. For sequentially generating the light sheet 1the illuminating laser beam is scanned in the x-direction, i.e.according to FIG. 3 out of the drawing plane or into the drawing plane,respectively. The laser beam may for example have a circularcross-section, but may in the alternative also be modulated in itscross-sectional shape, for instance have an oval cross-section, whereinthe longer axis of the oval cross-section extends in the x-direction.Likewise, the illumination beam path can for example be rectangular ormay have any other optional shape.

The SPIM-detection light beam path 11 extends in the z-direction, i.e.substantially perpendicular to the y-direction in which the illuminationbeam path 10 extends. It may be advantageous to deviate slightly fromthe perpendicular relationship between the illumination beam path andthe detection beam path, for instance smaller or larger angles than 90°between the two beam paths can be chosen, for instance for creatingbackground illumination for parts or particles within the object. It isalso possible to detect images from different angles and then combinethese, as for instance known as mSPIM technology. In the following, forkeeping the description simple, a rectangular relationship is described,but should be understood as also encompassing deviating angles which arehowever to be understood as more or less close to 90°. The detectionlight emanating from the object 2 either due to reflection orfluorescent light emission is collected by the objective 3. Particularlyfor multi-color detection a color filter 14 is provided downstream ofthe objective and is capable of filtering out detection light ofspecific wavelengths which is then directed via a tubular lens 15 to acamera 16, for instance a CCD-camera 16.

For generating a 3-dimensional image the object carrier 17 can bescanned in the z-direction for illuminating sequentially differentillumination planes within the object 2, each of the illumination planesbeing scanned by a scanned laser beam that is scanned in the x-directionand therefore illuminated by a sequentially formed light sheet 1 that issequentially formed in the respective illumination plane. A plurality ofin the z-direction adjacent sequentially formed light sheets 1 can becombined by “Rendering” in the z-direction.

In case 3-dimensional images should be generated by “Rendering” with az-drive, moving the object 2 has the advantage that the distance betweenthe respective adjacent illumination planes within the object and theobjective remains the same since neither the location of theillumination beam is changed nor the location of the camera 16. In thealternative it is possible to scan the light sheet 1 in z-direction, forinstance by a galvanometer. This requires though that the objective 3 isalso moved so that the distance between the respective illuminationplane and the object remains the same. In the alternative, it is ofcourse possible to move the entire SPIM detection optics comprising theobjective 3, the filter 14, the tubular lens 15 and the camera 16.

The illumination detection optics beam path 10 may as already mentionedfurther have the function of a confocal detection light beam path, forwhich purpose the detector 18 can be provided detecting light reflectedfrom the object in the y-direction and/or detecting emitted fluorescentlight. Simultaneously with image detection via the SPIM technologythrough the detection light beam path 11 it is also possible to performconfocal image detection in parallel since the light sheet 1 isgenerated sequentially by scanning in the x-direction. The confocallygenerated image is one dimension lower compared to the image detected bythe SPIM-detection. If for instance only a 2-dimensional image isgenerated by the SPIM-detection, i.e. an image within only one singleimage plane, it is also possible to detect a so called x-t image, i.e. a1-dimensional line image. This can for instance be used for determiningthe diffusion speed of specific molecules, that may be marked by amarker or are dyed for emitting fluorescent light, while the2-dimensional SPIM-image that is imaged simultaneously may providedifferent information, for instance information which molecules combineto which other molecules in the imaged imaging plane of the object.

The same applies if by means of the SPIM-detection a 3-dimensional imagewith a z-drive is generated, i.e. that a confocal 2-dimensional image isgenerated. In this fashion it is for instance possible to determinewhich molecules combine within the object in the 3-dimensional shapewith which other molecules, while in parallel the diffusion speed ofmolecules diffusing through specific planes can be determined. Thesimultaneous detection of a SPIM-image with the dimension “n” and aparallel confocally generated image with the dimension “n−1” allows incombination additional determinations, for instance the additional speedinformation allows to determine which individual elements, for examplemolecules or other elements, move within the object 2. In particular inthe field of microscope this finds new applications within livingorganisms.

FIG. 4 shows an additional embodiment of the invention. Similar elementsare designated with the same reference numerals as in the precedingfigures. Also in this figure the SPIM detection light beam path isdenoted by the reference numeral 11, while the illumination beam path isdesignated with the reference numeral 10. Also in this embodimentaccording to FIG. 4 the illumination beam path 10 has the function ofthe additional detection beam path wherein the signal detection isperformed by means of a photodiode 19 receiving through an aperture 20confocally detection light 21, and sends a respective signal via theimage processing unit 22 into a monitor 23. The detection light isdeflected by dichroic mirrors 24 to a photodiode 19. Instead of thephotodiode 19 also other light sensors, for instance Avalanche diodes,photomultipliers or a camera can be provided. Since this relates toconfocal image detection, no localization is necessary since theinformation about the location comes from the scanned illumination beam,i.e. it is known which image point of the object is illuminated at aspecific point in time, and therefore the information is known that thesignal received from that specific image point is from that image pointthat has been illuminated immediately prior to receiving the signal.Therefore, the expenditure for a camera is not necessary for confocaldetection.

For changing the focus of the sequentially generated light sheet 1 azoom lens 25 may be moved in the y-direction in relation to a furtherlens 26. For practical applications, a lens group will be provided forthis purpose, however, for simplification the discussed embodiment isdemonstrated with only 1 single zoom lens 25. The zoom optics 13 incombination with the illumination objective (macro objective) thereforeprovides an optical zoom allowing to modify the length of the focusrange 27. For simplification, the details of the SPIM-detection opticshave been omitted in FIG. 4 , as these optics have already been shown inFIG. 3 comprising the objective 3, the filter 14 and the tubular lens15. The schematically shown CCD-camera 16 provides for localizationstation in the x-y-plane for the detection light received in thez-direction. Via the SPIM image processing unit 28 the signals receivedby the CCD camera are processed and forwarded to the SPIM monitor 29. Ifa number of planes should be illuminated within the object 2 the objectcarrier 17 can be moved in the z-direction. As it can be clearlyunderstood from the illustration according to FIG. 4 the length Lmeaning the distance between the illumination plane and the CCD-cameraalways remains the same. As already explained in connection with FIG. 3, it is also possible to move the illumination beam in z-direction whilekeeping the object 2 at the same location, and by moving instead theCCD-camera, or as this might be easier to implement, to change theposition of the objective 3 in relation to the CCD camera 16.

FIG. 5 shows the embodiment illustrated in FIG. 4 , but as viewed from adifferent viewing point, namely in direction of the illumination beam,i.e. viewed in the y-direction. Illustrated are here in particular theindividual image points 30 which are scanned in the x-direction forgenerating a sequentially formed light sheet 1. The dichroic mirror 24deflects the detection light in direction of the photodiode 19. Theobject carrier 17 can be scanned in the z-direction, i.e. can be movedtowards the CCD-camera 16 or moved away from the CCD-camera forilluminating different illumination planes within the object.

In a further preferred embodiment, the light sheet 1 can be narrowedfurther by applying STED (Stimulated Emission Depletion), i.e. can bemade thinner for accomplishing a higher resolution in the z-direction.The basic structure of a STED microscope as it is known from the priorart is shown in FIG. 6 . An excitation beam 31 and a deactivation beam32 are combined by a beam combiner, in this embodiment for example by adichroic filter, to a joint beam path 34 that is directed through anobjective 3 onto the object 2. Several other optical elements can beprovided in between, for instance lenses, light conducting fibers orcolor filters. The deactivation beam is typically modulated in itsintensity distribution, which can for instance be accomplished by phaseplates, but also by lenses specifically structured for that purpose. Themodulation can be performed in that the deactivation beam has a zeropoint in the middle, meaning that the intensity is zero or very low inthe zero point while around this zero point a uniform, ring-shapedintensity maximum is provided. The dyes which are used in fluorescencemicroscopy react on specific excitation wavelengths and deactivationwavelengths allowing to excite fluorescence or to deactivatefluorescence specifically well. Typically, an excitation wavelength anda deactivation wavelength differ from each other and the excitation beamand the deactivation beam are sent in a time-delayed manner with respectto each other onto the object. By exciting, an image point of a specificsize can be excited to emit fluorescent light, while immediatelythereafter around the center of excitation a deactivation can beapplied, allowing to narrow down the fluorescent light emitted from thisimage point of the object 2 to a small image point and thereforeallowing to increase the resolution. For multi-color fluorescencemicroscopy a variety of different dyes can be used distinguishing fromeach other by different excitation wavelengths, i.e. excitation light ofa variety of wavelengths that allow a specifically strong excitation foremitting fluorescent light.

Preferably, the combination of dyes can be chosen such that these can bedeactivated by a common, same deactivation wavelength so that it can beavoided having to provide a variety of deactivation wavelengths.

The detection light 35, which is sent from the object back through theobjective 3, can be sent by a beam splitter, in this case likewise adichroic mirror 37, through a lens and a suitable aperture 38 foreliminating scattered light onto a photodetector 36.

Modulation of the deactivation beam 40 by a phase plate 39 isdemonstrated in FIG. 7 . Both the phase as well as the intensitydistribution can be modulated with such phase plates 39. For thispurpose, the phase plate is designed such that the deactivation beam 40is modulated, but not the excitation beam 41, i.e. the modulationdepends on the wavelength which differs for the deactivation beam 40 incomparison to the excitation beam 41.

It is to be understood that the STED-principle can also be implementedin the illumination beam path 10, as demonstrated in FIG. 4 . Inaddition to the laser 4 for the illumination light, which in this caseis equal to the excitation light, another laser can be provided forgenerating the deactivation light. An excitation beam and a deactivationbeam can either be combined into one beam path, as shown in FIG. 6 , orthe deactivation beam can be sent along a separate beam path. It wouldalso be possible to generate the excitation beam and the deactivationbeam with only one single laser 4 (white light laser). For this purpose,the beam is split to send one part of the beam through an AcoustoOptical Element as for instance an Acousto Optical Tunable Filter (AOTF)where the desired wavelength can be selected. By using pulsed lasers atime delay between the excitation beam and the deactivation beam can beimplemented by a so-called delay stage.

One embodiment of the invention is shown in FIGS. 8 a and 8 b . FIG. 8 ashows the cross-section of a circular excitation beam 41. The directionsx, y and z, as shown in FIGS. 4 and 5 , are also shown in the FIGS. 8 aand 8 b . An intensity distribution profile of the excitation beam isshown on the right side, and typically follows a Gaussdistribution-apart from relatively small side lobes due to diffraction.FIG. 8 b shows a deactivation beam 40 that is modulated such that itcomprises on both sides of the deactivation beam 41 an intensity maximumwith a zero point in between where the intensity is zero or at leastvery low. As it can be seen in FIG. 8 b , the intensity maxima of thedeactivation beam 40 which intensity maxima are provided on both sidesof the zero point do not have a circular cross-section, but comprise across-section having a flattened curvature towards the center incomparison to the side facing away from the excitation center. As thesequentially generated light sheet 1, which is demonstrated in FIG. 8 bin interrupted lines, only a small band in the center area remains inwhich the deactivation light does not deactivate fluorescence. At theright side next to the cross-section of the excitation beam 41,deactivation beam 40, and sequentially generated light sheet 1, theintensity distributions of the excitation beam 41 and the deactivationbeam 40 are demonstrated in FIG. 8 b.

The STED-deactivation beam can be selectively in addition turned on orit can be turned off, which can also be performed just per line or perpixel in x-direction. Since the information is known when thedeactivation beam is turned on or is turned off, the respective datastreams can be separated, i.e. into a first data stream for generatingan image based on the thicker light sheet and respectively covering alarger illumination area (compared to the cross-section of theillumination focus) and a larger illumination volume, and a second datastream for generating an image based on thinner light sheets with asmaller illumination area (in relation to the cross-section of theillumination focus) and a smaller illumination volume. This allows togenerate simultaneously in the z-direction a high resolution image byadding the STED-beam, and in z-direction an image of a lower resolutionwithout adding the STED-beam, but with the advantage of illuminating alarger volume within the object.

Independently of adding or turning off the STED-beam, this microscopeallows to generate in parallel simultaneously a SPIM-microscopy image ofthe dimension n and a confocally generated image of the dimension n−1,while these images can also be in parallel generated in the z-directionwith the high resolution or a lower resolution by additionally turningon or by turning off the STED-beam, respectively. In total, it ispossible to generate simultaneously 4 separate data streams generatingthe following sets of data:

i) 3-dimensional data set (SPIM) at a high resolution, but imaging asmaller volume of the object;

ii) 3-dimensional data set (SPIM) at a lower resolution, but imaging alarger volume of the object;

iii) 2-dimensional confocal image in z-direction at a high resolution;and

iv) 2-dimensional confocal image in z-direction at a lower resolution.

If only one plane should be illuminated with the light sheet, thefollowing images can be generated simultaneously:

i) 2-dimensional image (SPIM) at a high resolution, but imaging asmaller volume of the object;

ii) 2-dimensional image (SPIM) at a lower resolution, but imaging alarger volume of the object;

iii) 1-dimensional confocal image x-t in z-direction at a highresolution; and

iv) 1-dimensional confocal image x-t in z-direction at a lowerresolution.

The number of light sheets in z-direction is not dependent on thex-dimension, i.e. it is possible to choose from different image formats,for instance the number of pixels in x-direction of 512, while thenumber of pixels in the z-direction can be more or less. For obtaining acontinuous data set in the z-direction without gaps, the feed motion inz-direction must be chosen such that always some overlap is guaranteed(Nyquist Theorem).

FIG. 9 shows an additional embodiment of the invention where the samecomponent parts in comparison to the embodiment according to FIG. 3 aredenoted with the same reference numerals. In addition, an illuminationobjective 42 is provided that can be provided in addition to the zoomoptics 13. The illumination objective can also be part of the opticalzoom, as for instance in the example according to the embodiment shownin FIG. 4 .

In contrast to the illumination with a continuous laser (continuous waveCW) also a pulsed laser can be used for multiphoton fluorescencemicroscopy sending exciting photons of a long wavelength and of arelatively low energy which is therefore specifically suitable foravoiding damage to the sample, which may particularly be important forbiological samples. Like the detection of the SPIM-signal also amultiphoton signal can be extracted by means of a switchable mirror 43from the detection beam path 44 extending in the z-direction and can bedetected by a photomultiplier or an Avalanche photodiode 45. Other thanthat, the SPIM-signal can be detected by the camera 16 as alreadydescribed with reference to FIG. 3 .

If the camera 16 works fast enough, as a further variation of theembodiment shown in FIG. 9 , the switchable mirror 43 can be dispensedwith and instead the camera 16 can be used for detecting the multiphotonsignal. Since in case of a multiphoton illumination together withdetecting the signal in z-direction, i.e. perpendicular to the directionof illumination, for each illumination point on the object a line isimaged onto the camera 16, and by scanning in the x-direction,sequentially a scan-line on the object that extends in the x-direction,an area is sequentially illuminated on the camera 16 that needs to bereduced by software reducing the data back to a line. By scanning inz-direction sequentially several lines of the object can be detected andfurther processed to an image of an area of the object.

In simple words, this variation of the SPIM signal detection structureis used for a multiphoton signal detection, which is possible with fastcameras, for instance cameras that can detect up to 1000 images persecond in the format 512×512.

A specific advantage of the SPIM signal detection structure for themultiphoton signal detection is a significantly increased signalstrength, among other reasons due to the following reasons: Forgenerating light sheets typically illumination objectives of a lownumerical aperture are used, for instance objectives with a numericalaperture in the range of 0.04 NA. If now the SPIM detection path is usedfor detecting generated signals from the multiphoton illumination,significantly higher signal strengths can be obtained since the usedobjectives provide for a much higher numerical aperture (NA) (forinstance 1.0 NA). This means gaining signal strength by more than afactor 20.

If the SPIM detection beam path (in z-direction) is used for detectingfluorescent light and the light is sent to a photomultiplier or an APDor APD array or according to the variation described above directly sentto a fast camera, a much higher efficiency can be reached as by usingthe illumination optics for signal detection in the y-direction, sincetypically the used numerical aperture of the illumination system islower than the numerical aperture of the detection system of the SPIMarrangement.

Using multiphoton detection has several advantages. Almost exclusivelyonly those fluorochromes are excited that are in the focus, sinceexcitation requires several photons to arrive more or lesssimultaneously, which happens almost exclusively in the focus, or put inother words, the likelihood of exciting outside the focus is very low.Another advantage is the higher penetration depth when using wavelengthsin the IR range (scattering is low). Another advantage is that nopinhole is necessary, since the entire emitted light can be allocated tothe illumination focus. This allows also collecting light from alldirections. In contrast, confocal microscopes require that scattered anddeflected light needs to be suppressed, which reduces the signalstrength. Apart from all these advantages in having a higher signalstrength the illumination intensity is lower and therefore avoids damageto the sample, while this solution further provides the structuraladvantage that the already provided for detection optics of theSPIM-microscope can be used for the multiphoton signal detection,allowing all these advantages without further structural expenditure andonly relatively low expenditure on software.

The STED-technology can further also be used for the multiphotonillumination, allowing reducing a resolution of about 300 nm in themultiphoton mode to be reduced to a resolution of only a few nm in themultiphoton-plus-STED mode.

From a software perspective, it is also possible to separate the datadetected by the camera 16 into lines and pixels and to generatesimultaneously a SPIM image and a multiphoton image. This allowsdispensing with switching between SPIM mode and multiphoton mode, or inthe alternative the simultaneous operation in the SPIM mode and in themultiphoton mode can be added as a switching option.

Summarizing, the microscope according to the invention applies a partialaspect of confocal microscopy, namely the partial aspect of theillumination optics, for generating an image based on theSPIM-technology, wherein the illumination optics is further modified toa zoom optics, and according to the invention further a confocal imagecan be generated that is one dimension lower than the image generated bythe SPIM technology, or in the alternative in addition to the confocalimage a multiphoton image can be generated, also in lieu of the imagegenerated by the SPIM technology. This does not only allow to influencethe image generated by the SPIM technology much more flexibly but allowsalso for additional image information obtained confocally or bymultiphoton detection, and depending on the specific application, theresulting images can also be combined as an overlay with the SPIM and/orthe multiphoton images. By adding or by turning off a STED beam theimage can be influenced further and it is possible to generate evenadditional data streams for a variety of images, which further have theadvantage of being generated simultaneously. This allows for providingthe microscope with a manifold utility with synergistic effects in thepossibilities of modulating the image and at the same time in the numberof analyzable image information.

What is claimed is:
 1. A SPIM-microscope comprising: a light sourcesending an illumination light beam from a y-direction onto an object tobe imaged; a camera detecting in a z-direction as a first detectiondirection light emanating from the object as at least one of fluorescentlight and reflected light, wherein the z-direction extends substantiallyperpendicular to the y-direction; an x-scanner generating a sequentiallight sheet by scanning the illumination light beam in an x-direction,wherein the x-direction extends substantially perpendicular to they-direction and to the z-direction and the light sheet is sequentiallyformed in a plane that is defined by the x-direction and they-direction; an illumination optics comprising a zoom optics provided ina beam path of the illumination light beam, the zoom optics beingadapted to change the focal length of the illumination light beam andadapted to detect a larger area of the object by sequentially detectingsequences of images along the y-direction that have an increasedresolution along the z-direction; and an image processing unit thatcombines these sequences of images by image stitching into one largeoverall image.
 2. The SPIM-microscope according to claim 1, furthercomprising a photodetector detecting detection light sent from theobject in the opposite direction of the y-direction as a seconddetection direction, the detection light being at least one offluorescent light and reflected light.
 3. The SPIM-microscope accordingto claim 2, wherein in parallel to the 2-dimensional wide field imagegenerated by the SPIM-technology also confocally a 1-dimensional imageof the object is generated that comprises a line extending in thex-direction.
 4. The SPIM-microscope according to claim 1, furthercomprising a first z-scanner moving the object in the z-direction sothat sequentially a plurality of light sheets spaced in z-direction withrespect to each other are generated in the respective illuminationplanes, wherein a distance between the respective light sheets and thecamera remains unchanged.
 5. The SPIM-microscope according to claim 4,further comprising an image processing unit that combines thesequentially generated images correlating to the respective plurality oflight sheets which are spaced in the z-direction in a distance to eachother to a 3-dimensional image.
 6. The SPIM-microscope according toclaim 5, further comprising a photodetector detecting detection lightsent from the object in the opposite direction of the y-direction as asecond detection direction, the detection light being at least one offluorescent light and reflected light, wherein in parallel to the3-dimensional image generated by the SPIM-technology also confocally a2-dimensional image of the object is generated in a plane defined by thex-direction and the z-direction.
 7. The SPIM-microscope according toclaim 1, further comprising: a second z-scanner moving the illuminationlight beam in the z-direction in relation to the object for generatingsequentially a plurality of light sheets spaced in the z-direction withrespect to each other; and a third z-scanner that tracks the detectionbeam path according to the deflection of the illumination light beam inz-direction by the second z-scanner so that a distance between the lightsheet and the camera remains unchanged.
 8. The SPIM-microscope accordingto claim 7, further comprising an image processing unit that combinesthe sequentially generated images correlating to the respectiveplurality of light sheets which are spaced in the z-direction in adistance to each other to a 3-dimensional image.
 9. The SPIM-microscopeaccording to claim 8, further comprising a photodetector detectingdetection light sent from the object in the opposite direction of they-direction as a second detection direction, the detection light beingat least one of fluorescent light and reflected light, wherein inparallel to the 3-dimensional image generated by the SPIM-technologyalso confocally a 2-dimensional image of the object is generated in aplane defined by the x-direction and the z-direction.
 10. TheSPIM-microscope according to claim 1, further comprising zoom opticsthat are adapted to vary the focal length of the illumination light beamis an optical zoom having lens groups which are mechanically moved withrespect to each other.
 11. The SPIM-microscope according to claim 10,wherein the zoom optics is adapted to expand or shorten the focal lengthof the illumination light beam by changing the numerical aperture andtherefore expand or shorten the length of the field in they-illumination direction that is illuminated by the light sheet.
 12. TheSPIM-microscope according to claim 1, further comprising an electroniczoom by which the scanning length in the z-direction can be changedalong which a plurality of spaced apart light sheets are generatedsequentially.
 13. The SPIM-microscope according to claim 1, wherein thenumber of light sheets which are spaced with respect to each other inthe z-direction is maintained unchanged by the electronics zoomindependently of the scanning length in the z-direction within theobject that has been selected.
 14. The SPIM-microscope according toclaim 1, wherein the illumination light beam scanned in the x-directionis turned off at or close to the return point where the maximum scanninglength in x-direction has been reached.
 15. The SPIM-microscopeaccording to claim 1, wherein the camera is an area detector chosen fromthe group consisting of CMOS-camera, CCD-camera or array-detectors. 16.The SPIM-microscope according to claim 1, further comprising a switchadapted to switch between the following operational modes: confocaldetection of detection light opposite to the y-illumination direction;SPIM-detection of wide field detection light in the z-direction; andsimultaneous detection of the aforementioned confocal detection andSPIM-detection.
 17. The SPIM-microscope according to according to claim1, further comprising a deactivation light source sending from they-direction a deactivation light beam onto the object making thesequentially generated light sheet in the z-direction thinner, whereinthe deactivation light beam is sent offset in the z-direction onto theobject in relation to the illumination light beam and extends inparallel to that illumination light beam that is scanned in thex-direction.
 18. The SPIM-microscope according to claim 17, furthercomprising an excitation light beam modulator adapted to modulate theexcitation light beam into a Bessel beam.
 19. The SPIM-microscopeaccording to claim 1, wherein the illumination light source is a pulsedlaser; the illumination light beam is a multiphoton laser beam; and amultiphoton signal is detected in the z-direction.
 20. TheSPIM-microscope according to claim 19, wherein the camera is a fastcamera that is adapted to detect in addition to the SPIM signal also themultiphoton signal.
 21. The SPIM-microscope according to claim 19,further comprising a switchable mirror allowing extracting themultiphoton signal from the z-direction and directing it to aphotodetector.
 22. The SPIM-microscope according to claim 1, wherein alarge area of an object is imaged by combining sequentially imagedadjacent object areas.
 23. The SPIM-microscope according to claim 1,wherein a usable size of the light sheet is decreased by increasing anumerical aperture by the zoom optics.
 24. The SPIM-microscope accordingto claim 1, wherein a focal length of the illumination light beam isexpanded or shortened by changing a numerical aperture by the zoomoptics in combination with the illumination optics, allowing expandingor shortening a length of a field that is illuminated by the light sheetin the y-illumination direction.
 25. The SPIM-microscope according toclaim 1, wherein the object is moved in the x-direction or in they-direction and a new area of the object is illuminated and images aredetected by the camera.